Expression of GFPuv and mCherry in transformed <i>E. chaffeensis</i>.

<p>Himar1 transposon mutants of <i>E. chaffeensis</i> were generated following electroporation of the constructs into the cell-free organisms recovered from infected ISE6 tick cells and re-cultured in macrophage and tick cells. A drop of culture suspension under a cover slip was imaged under uv-illumination using an Olympus BX61 spinning disk confocal microscope and a Qfire digital camera. Panels A and B represent <i>E. chaffeensis</i> Arkansas isolate expressing mCherry and GFPuv, respectively. Bars = 10 µm.</p

Clonal isolation of the <i>E. chaffeensis</i> mCherry and GFP mutants.

<p>Host cell-free organisms recovered from nearly 90% infected culture flask were used to make 10-fold serial dilutions and cultured in DH82 cultures. The culture flasks resulting in <i>E. chaffeensis</i> growth from the highest dilution were used to isolate genomic DNA and for assessing the Himar1 insertions by performing insertion-specific PCRs. Lanes 1–8, genomic DNA from mixed populations of mutants prior to cloning by serial dilution was used as the template for PCRs targeting to the 8 Himar genomic insertion sites (as listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003171#ppat-1003171-g003" target="_blank">Figure 3</a>); Lanes 1c–8c, as in lanes 1–8, but using DNAs recovered after serial dilution cloning of the organisms; Lanes with the letter N refer to no template DNA controls for each of the PCRs and lane M contained the 1 kb+ DNA molecular weight marker.</p

Nested PCR verification of the <i>E. chaffeensis</i> infection status and for the transposon insertion sites assessed in deer blood DNA^#.

#<p>Second infection experiment;</p>**<p>Genomic insertion sites as defined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003171#ppat-1003171-g003" target="_blank">Figure 3</a>.</p

Verification of the <i>E. chaffeensis</i> infection status by culture and for the transposon insertion sites in mCherry mutant infected deer blood^#.

#<p>First infection experiment.</p>*<p>Blood-derived buffy coats or DNAs from the first infection experiment were assessed for the presence of mutants by culture recovery and nested PCR, respectively. Culture positives refer the presence of organisms as inclusions in the DH82 cells as determined by cytospin and polychromatic stained slides. Blood recovered on days 5, 32, 35, 42, 46, 49 post-infection were also assessed and tested negative for the viable pathogen or predicted amplicons. The − and + signs refer to samples tested negative or positive.</p>**<p>Genomic insertion sites as defined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003171#ppat-1003171-g003" target="_blank">Figure 3</a>.</p

Transcriptional analysis of the transposon mutants with insertions within the protein coding regions of Ech_0379, Ech_0601, Ech_0660 and Ech_0230 genes.

<p>Total RNA isolated from wild type <i>E. chaffeensis</i> grown in DH82 culture (lanes 1, 2, 9, 10, 17,18, 25, and 26) and AAE2 tick cell culture (lanes 3, 4, 11, 12, 19, 20, 27, and 28) and the mCherry mutant culture derived (lanes 5,6, 29, and 30) and GFPuv first transformation (lanes 13 and 14) and second transformation (lanes 21 and 22) cultures grown in DH82 cultures was assessed by RT-PCR for evaluating the gene expression. PCR products resolved in lanes 1–8 included primers targeted to the Ech_0379 and lanes 9–16 contained primer set for the gene Ech_0601, lanes 17–24 contained primers targeted to Ech_0660, and lanes 25–32 had primers targeted to Ech_0230. (Lanes 2, 4, 6, 10, 12, 14, 18, 20, 22, 26, and 28 did not include reverse transcriptase; lanes 7, 15, 23, and 31 served as the positive controls as they included the wild type genomic DNA; and lanes 8, 16, 24, and 32 contained no template and served as negative controls.).</p

Nested PCR verification of the <i>E. chaffeensis</i> infection with clonal population mutants in deer blood.

*<p>Clonal population mutants 2 (mutation in the noncoding region between the genes Ech_0284 and Ech_0285) and 9 (mutation in the gene Ech_0660) as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003171#ppat-1003171-g003" target="_blank">Figure 3</a>.</p

A cartoon illustration of the <i>E. chaffeensis</i> genomic locations mapped for the transposon mutants.

<p><i>E. chaffeensis</i> genomic DNA from three independent transformations with mCherry (one transformation) and GFPuv (two transformations) Himar1 transposon plasmids was used to determine the integration locations by inverse PCRs and ST-PCRs followed by DNA sequence analysis. Genomic locations of the insertion sites and the genes at or near the insertions, as per the whole genome data (GenBank # CP000236.1), were presented. The gene expression data assessed by RT-PCR were also included in the figure (E, expressed gene; m, in macrophage culture; t, in tick cell culture; No, gene not expressed). The insertions in mCherry transformants are shown as solid red bars, and insertions in GFPuv transformants are depicted as solid green bars (dark green bar, the first GFPuv transformants; light green bar, the second GFPuv transformant).</p

Schematic representation of the preparation of <i>E. chaffeensis</i> transposon mutants and <i>in vivo</i> screening to identify genes important for the pathogen's growth in deer and tick hosts.

<p>The protocol involves recovering <i>in vitro</i> culture-derived <i>E. chaffeensis</i> organisms, subjecting them to transposon mutagenesis, reinfecting to naïve host cells, selecting the mutants in culture resisting to antibiotic clearance, infecting a natural host (white-tailed deer), acquisition feding of ticks on the deer and finally assessing the mutants survived in deer and ticks.</p

Basic Local Alignment Search Tool (BLAST) homology search analysis of Ech_0379.

<p>The translated protein coding sequence of Ech_0379 was subjected to BLAST analysis by searching against protein databases available through the National Center for Biotechnology Information (NCBI) web site. The homologies having significant sequence similarities with identities above 30% and positives above 50% to a sequence motif in the Ech_0379 were identified and presented. Three significant hits were presented in the Figure.</p

Southern blot analysis of Himar1 transformants of <i>E. chaffeensis</i>.

<p>Genomic DNA from Himar1 transposon <i>E. chaffeensis</i> mutants was assessed by DNA blot analysis using a spectinomycin resistance gene (<i>aad</i>) probe following digestions with different restriction enzymes (N, Nde I; S, Spe I; Bs, Bsrg I; E, EcoR V; B, Bgl II and Hind III). A) <i>E. chaffeensis</i> genomic DNA was recovered from the mutant organisms of mCherry plasmid transformed (m); GFPuv plasmid transformed (1^st experiment) (g) grown in ISE6 tick cells (I) or DH82 macrophages (D). B) Genomic DNAs from the mCherry and GFPuv (1^st experiment) mutants propagated in DH82 cultures was also assessed at two different randomly selected harvest times (separated about 1 month apart) to evaluate the stability of the transformants over time. The lanes mD1 and mD2 represent two different days when mCherry mutants were harvested, and gD1 and gD2 represent different harvest dates for GFPuv mutants. C) Second GFPuv plasmid transformed mutants grown in DH82 culture and assessed by DNA blot analysis (GD). Genomic locations for the DNA fragments indicated by the arrows were established by sequence analysis. Values shown on the left side of panels represent DNA size markers.</p